title: Lanthanide chelate complementation and hydrolysis enhanced luminescent chelate in real-time reverse transcription polymerase chain reaction assays for KLK3 transcripts.
abstract: The requirement for high-performance reporter probes in real-time detection of polymerase chain reaction (PCR) has led to the use of time-resolved fluorometry of lanthanide chelates. The aim of this study was to investigate the applicability of the principle of lanthanide chelate complementation (LCC) in comparison with a method based on hydrolysis enhancement and quenching of intact probes. A real-time reverse transcription (RT) PCR assay for kallikrein-related peptidase 3 (KLK3, model analyte) was developed by using the LCC detection method. Both detection methods were tested with a standard series of purified PCR products, 20 prostatic tissues, 20 healthy and prostate cancer patient blood samples, and female blood samples spiked with LNCaP cells. The same limit of detection was obtained with both methods, and two cycles earlier detection with the LCC method was observed. KLK3 messenger RNA (mRNA) was detected in all tissue samples and in 1 of 20 blood samples identically with both methods. The background was 30 times lower, and the signal-to-background (S/B) ratio was 3 times higher, when compared with the reference method. Use of the new reporter method provided similar sensitivity and specificity as the reference method. The lower background, the improved S/B ratio, and the possibility of melting curve analysis and single nucleotide polymorphism (SNP) detection could be advantages for this new reporter probe.
authors.affiliation: Division of Biotechnology, University of Turku, 20520 Turku, Finland. Electronic address: saeid.alinezhad@utu.fi.
authors.forename: Saeid
authors.initials: S
authors.lastname: Alinezhad
authors.forename: Riina-Minna
authors.initials: RM
authors.lastname: Väänänen
authors.forename: Ari
authors.initials: A
authors.lastname: Lehmusvuori
authors.forename: Ulla
authors.initials: U
authors.lastname: Karhunen
authors.forename: Tero
authors.initials: T
authors.lastname: Soukka
authors.forename: Esa
authors.initials: E
authors.lastname: Kähkönen
authors.forename: Pekka
authors.initials: P
authors.lastname: Taimen
authors.forename: Kalle
authors.initials: K
authors.lastname: Alanen
authors.forename: Kim
authors.initials: K
authors.lastname: Pettersson
mesh_terms: D002614:Chelating Agents / Q000737:chemistry; D005260:Female; D005456:Fluorescent Dyes / Q000737:chemistry; D006801:Humans; D006868:Hydrolysis; D007610:Kallikreins / Q000097:blood / Q000235:genetics*; D028581:Lanthanoid Series Elements / Q000737:chemistry; D057230:Limit of Detection; D008297:Male; D015394:Molecular Structure; D020641:Polymorphism, Single Nucleotide / Q000235:genetics; D017430:Prostate-Specific Antigen / Q000097:blood / Q000235:genetics*; D011471:Prostatic Neoplasms / Q000097:blood / Q000235:genetics*; D012333:RNA, Messenger / Q000097:blood / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D013997:Time Factors; D014158:Transcription, Genetic / Q000235:genetics*
chemical_list: D002614:Chelating Agents; D005456:Fluorescent Dyes; D028581:Lanthanoid Series Elements; D012333:RNA, Messenger; C549870:KLK3 protein, human; D007610:Kallikreins; D017430:Prostate-Specific Antigen
keywords: Lanthanide chelates; Reverse transcriptase quantitative polymerase chain reaction; Time-resolved fluorometry